JP5453343B2 - Method for producing polypeptide in Aspergillus mutant cells - Google Patents

Abstract

A method is provided for producing a polypeptide of interest by (a) cultivating a mutant of a parent Aspergillus cell, wherein (i) the mutant comprises a first nucleic acid sequence encoding the polypeptide and a second nucleic acid sequence comprising a modification of at least one of the genes responsible for the biosynthesis or secretion of at least one toxin, and (ii) the mutant produces less of the toxin than the parent Aspergillus cell when cultured under the same conditions; and (b) isolating the polypeptide from the culture medium. Also, mutants of Aspergillus cells are provided, as well as methods for obtaining the mutant cells.

Description

  The present invention relates to a method for producing a polypeptide of interest in a toxin-deficient Aspergillus mutant cell. The present invention also relates to mutants of Aspergillus cells and methods for obtaining said mutant cells.

BACKGROUND OF THE INVENTION Recently, the commercial value of utilizing recombinant host cells for the expression of heterologous (heterologous) polypeptides that can only be obtained in small amounts by other methods or only by purification from natural sources. Mass production of certain polypeptides, such as industrially important enzymes and secondary metabolites, has been greatly simplified. Currently, there are a variety of expression systems selected for the production of a particular polypeptide, including eubacteria and eukaryotic hosts. The selection of an appropriate expression system often depends not only on the ability of the host cell to produce a polypeptide having the desired composition and conformation, but also often depends on the intended end use of the protein.

  One problem encountered with the use of certain host systems is mycotoxin production. Many fungi used as host cells in the production of a polypeptide of interest possess genes that encode enzymes involved in the biosynthesis of various toxins. For example, cyclopiazonic acid, kojic acid, 3-nitropropionic acid and aflatoxins are known toxins produced, for example, in Aspergillus flavus. Similarly, trichothecenes are produced in many fungi, such as Fusarium species, such as Fusarium venenatum and Trichoderma species. A detailed review of toxin formation in various fungi can be found in the Handbook of Toxic Fungal Metabolites, Richard J. Cole & Richard H. Cox, Academic Press, 1981.

  The formation of such toxins that occur during fermentation of the polypeptide of interest is highly undesirable because they can be detrimental to the health and environment of both workers and users.

  Therefore, great efforts are being made to ensure that such toxins are not formed in quantities that would affect health under the conditions used for the production involved. This is mainly done by a large-scale analysis program by analyzing toxins directly or by bioassay and / or dietary experiments. In many cases, such large programs are run once per production, affecting both the cost of production and the time to sell the product.

(Also referred to hereinafter as "CPA") cyclopiazonic acid is a weak acid (pK _a: 3.5) precipitates under acidic conditions. It forms a metal chelate, which is separated by dilute acid. CPA is highly toxic because it causes degenerative lesions and necrosis, especially in many organs, and selectively inhibits Ca ²⁺ -ATPase. CPA is produced in alpha and beta forms, which are precursors of the alpha form. CPA is produced by the genus Aspergillus, but is also produced by other fungi, such as the genus Penicillium.

Kojic acid (hereinafter also referred to as “KA”) is produced by a number of Aspergillus species, but is also produced by other fungi such as Penicillium and some other bacteria. It is weakly alkaline (pK _a : 7.9; phenol group) and forms complexes with many metal ions. It has antibacterial activity and is less toxic to animals. It is a precursor to many synthetic compounds such as insecticides and pigments.

3-Nitropropionic acid (hereinafter also referred to as “3-NPA”) is a natural nitro compound. It is a class of fungi, especially Aspergillus [A. A. flavus , A. flavus Wenty ( A. wentii )] and Penicillium [P. Produced by P. atroventum . Production in several bacteria has also been reported. This acid and its esters have also been found in some plants. It is rather toxic in itself because it causes, for example, anemia. It can also be partially converted to nitrite, another toxic compound, in the gastrointestinal tract. 3-Nitropropionic acid affects the Krebs cycle by irreversibly inhibiting succinate dehydrogenase and reversibly inhibiting isocitrate lyase, fumarase and aspartase.

Aflatoxins are highly biologically active and are secondary metabolites produced by the fungi Aspergillus flavus Link ex. Fries and Aspergillus parasiticus Speare; RW Detroy et al., “Aflatoxin and related compounds ", Microbial Toxins, Volume 6 (A. Ciegler, S. Kadis and SJ Ajl), Academic, New York, 1971, pages 3-178. The major aflatoxins are B ₁ , B ₂ , G ₁ and G ₂ . This metabolite is particularly aflatoxin B ₁ represents not only toxic to animals and humans, even the most carcinogenic of all known natural compounds.

  Malformin and ochratoxin are Produced by A. niger.

  By eliminating or reducing the host organism's ability to produce toxins, the formal approval procedure will be much simpler and the analytical program can be reduced, thus reducing time and costs in the production phase.

  Currently, there is a need for toxin-deficient Aspergillus mutant cells (ie, safe organisms that are preferably classified as GRAS) that are suitable for producing the polypeptide of interest in an efficient and economical manner. The present invention fulfills this need by providing production of a polypeptide of interest using a toxin-deficient Aspergillus mutant host cell and by providing a method for producing such a mutant host cell. There is also a need to provide Aspergillus mutant cells that have one or more toxin genes that are not expressed, ie, silent genes.

  According to one aspect of the present invention, a method of producing a polypeptide of interest by an Aspergillus mutant host cell, comprising: (a) culturing a mutant of a parent Aspergillus cell, wherein (i) the mutant is A first nucleic acid sequence encoding a polypeptide of interest, and a second nucleic acid sequence comprising at least one genetic alteration responsible for biosynthesis or secretion of at least one toxin, and ( ii) providing a method wherein said variant produces less toxin than parental Aspergillus cells when cultured under the same conditions; and (b) isolating said polypeptide from the medium.

  In a preferred embodiment of the invention, the mutant Aspergillus cells produce at least 90% less toxin than the parental cells when cultured under the same conditions. Preferably, the mutant produces less one or more of cyclopiazonic acid, kojic acid, 3-nitropropionic acid and aflatoxin than the parent Aspergillus cells when cultured under the same conditions.

  According to another aspect of the invention, a toxin-deficient Aspergillus mutant host cell useful for the production of a heterologous polypeptide of interest, wherein the toxin is at least one toxin as compared to the Aspergillus parent cell when cultured under the same conditions. Cells are provided that have been genetically altered to produce small amounts of.

The Aspergillus cell of the present invention is preferably A. A. oryzae , A. oryzae A. aculeatus , A. Nidulans , A. Nidulans A. ficuum , A. A. flavus , A. flavus Fetidas ( A. foetidus ), A. A. soja , A. A. sake , A. sake Niger, A. niger Japonica ( A. japonicus ), A. japonicus Parasitics ( A. parasiticus ) and A. Selected from the group consisting of phoenix ( A. phoeicus ).

  In another aspect of the invention, a method for obtaining a toxin-deficient Aspergillus mutant host cell comprising: (a) a first nucleic acid sequence encoding a polypeptide of interest in the Aspergillus parent host cell and at least one Introducing a second nucleic acid sequence comprising an alteration of at least one gene responsible for biosynthesis or secretion of one toxin; and (b) from step (a) said first and second nucleic acid sequences There is provided a method comprising identifying a variant comprising.

In one aspect, the present invention relates to Aspergillus mutant cells suitable for expression of heterologous polypeptides from which one or more silent toxic genes have been removed.
These and other aspects will be described in more detail in the description below.

FIG. 1 shows an Aspergillus oryzae DCAT-S performed using restriction enzymes EcoRI, SalI, BbuI, XhoI and XbaI using a 1 kb ³² P-labeled DNA BglII fragment from pJaL499 containing the DCAT-S gene as a probe. The genome restriction map of a gene is shown. FIG. 2 shows the construction of the disrupted plasmid p (DCAT-S-pyrG).

DETAILED DESCRIPTION OF THE INVENTION Definitions For purposes of this application, the following terms are defined for a better understanding of the invention.
The term “vector” refers to a plasmid, cosmid, phage or any other mediator that allows the insertion, propagation and expression of a gene or DNA sequence encoding a polypeptide of interest (including precursor forms thereof).

  The term “host” means any cell that will allow the expression of the polypeptide of interest, including its precursor form.

The term “transformation” refers to an uptake that allows expression of a heterologous DNA sequence by a cell.
The term “mutant” host (or strain) refers to a genetically altered strain that encompasses both mutants and transformants of a wild type strain.

  The term “toxin” refers to a fungal metabolite having phytotoxicity, animal toxicity and antibiotic activity. The term “mycotoxin” is generally defined as a secondary metabolite produced by a fungus that has the potential to cause adverse health hazards to humans and animals at the level of exposure. In the present specification, “toxin” and “mycotoxin” can be used interchangeably.

  The term “toxin deficiency” as used for the mutant cells of the present invention means that the mutant has incomplete toxin production compared to its parent strain, ie, the mutant has at least one, preferably It means that multiple toxins are produced in a smaller amount than the parent cell.

  The term “same conditions” as used in step a) of the method of the invention relates to the toxin production of the mutant relative to the toxin production of the parent cell, and the same for fermentation of the mutant and the parent strain, eg for pH, temperature, oxygen etc. Used to indicate that a condition is used. Parental and mutant cells can be compared for production of the toxin in question under conditions that produce the polypeptide of interest, or under conditions that produce one or more toxins.

The present invention relates to a variant of an Aspergillus host cell useful for the expression of a polypeptide of interest that has been genetically altered to express a significantly reduced level of one or more toxins compared to the parent cell. Provide the body. The host cell is derived from the parent cell, but may be a wild type cell.

  The host strain can be any Aspergillus host cell conventionally used for expression of the polypeptide of interest.

In a preferred embodiment, the Aspergillus host cell useful for the production of the polypeptide of interest is, Aspergillus subgroups Yurochiumu (Eurotium) [e.g. A. Represented by A. restrictus species], Chaetosartorya [e.g. A. restrictus species]. Represented by A. cremeus species], Sclerocleista [e.g. A. cremeus species]. Represented by A. ornati species], Satoia [eg A. ornati species]. Niger (A. niger) is represented by the species], Neosartorya (Neosartorya) [e.g. A. A. fumigatus A. fumigatus Represented by the species A. cervinus ], Hemicarpenteles [eg A. cervinus ] Kurabatsusu (A. clavatus) represented by the seed], Petoromisesu (Petromyces) [e.g. A. A. flavus , A. flavus A. candidus , A. candidus Typified by A. sparsus species), emerisera (eg, represented by A. nidulans , A. versicolor , A. ustus species), and Feneria (Fenellia) [e.g. A. terreus (A. terreus) typified by the species] selected from the group consisting of.

In a particularly preferred embodiment, the Aspergillus host cell is Orize, A. Accurators, A. Ficuum, A.H. Fraves, A.M. Fetidas, A. Soya, A. Salmon, A. Niger, A. Nidurance and A.M. Selected from the group consisting of Japonicas. Among them, Aspergillus oryzae, Aspergillus niger, A. Parasitics and A.I. Phoenix is most preferred.
The above examples of host cells of the invention are named according to the currently accepted biotaxonomy.

Toxin A toxin to be reduced or eliminated in production according to the present invention is any toxin produced by Aspergillus or encoded by a gene owned by Aspergillus but not necessarily expressed. Also good. For example, the aflatoxin gene is A. It is known to be present in oryzae but not expressed from this species. However, it may still be advantageous to remove aflatoxin pathway genes even if they are not expressed. This is described below and illustrated in Example 6.

  In particular, the toxin to be reduced or eliminated is cyclopiazonic acid (CPA), such as its alpha or beta form, kojic acid (KA), 3-nitropropionic acid (NPA), emodin, malformin (eg malformin A or B). , Aflatoxin, ochratoxin, flavioline and secaronic acid (eg secaronic acid D). For a description of these toxins, see the Background section of the invention herein and the Handbook of Toxic Fungal Metabolites mentioned therein.

Dimethylallyl-cycloacetoacetyl-L-triprofan synthase (DCAT-S) of the present invention
The present invention relates to (a) dimethylallyl-cycloacetoacetyl-L-tryptophan synthase having the amino acid sequence of SEQ ID NO: 2; (b) an allelic variant of (a); and (c) dimethylallyl-cycloacetoacetyl- It also relates to an isolated dimethylallyl-cycloacetoacetyl-L-tryptophan synthase obtained from a filamentous fungus selected from the group consisting of (a) or (b) fragments having L-tryptophan synthase activity.

  Preferably, the dimethylallyl-cycloacetoacetyl-L-tryptophan synthase of the present invention comprises the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof. In a more preferred embodiment, the dimethylallyl-cycloacetoacetyl-L-tryptophan synthase of the present invention comprises the amino acid sequence of SEQ ID NO: 2. In another preferred embodiment, the dimethylallyl-cycloacetoacetyl-L-tryptophan synthase of the invention has the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, wherein said fragment is dimethylallyl-cycloacetoacetyl-L-tryptophan Has synthase activity. The fragment of SEQ ID NO: 2 is a polypeptide in which one or more amino acid sequences have been deleted from the amino terminus and / or carboxy terminus of this amino acid sequence. In the most preferred embodiment, dimethylallyl-cycloacetoacetyl-L-tryptophan synthase has the amino acid sequence of SEQ ID NO: 2.

  Preferably, the fragment of SEQ ID NO: 2 has at least 320 amino acid residues, most preferably at least 350 amino acid residues.

  Allelic variant refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs naturally through mutation and can cause phenotypic polymorphism within a population. The genetic variation may be silent (no change in the encoded polypeptide) or may encode a polypeptide having an altered amino acid sequence. The term allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

The amino acid sequence of SEQ ID NO: 2 or a partial sequence thereof can be used to design an oligonucleotide probe, ie a nucleic acid sequence encoding the dimethylallyl-cycloacetoacetyl-L-tryptophan synthase of the present invention, eg, in the art Can be used to identify and clone DNA encoding dimethylallyl-cycloacetoacetyl-L-tryptophan synthase from another filamentous strain using the nucleic acid sequence of SEQ ID NO: 1 or a partial sequence thereof. In particular, such probes can be used for hybridization with genomic DNA or cDNA of the genus or species of interest according to standard Southern blotting techniques to identify and isolate the corresponding gene. Such probes can be much shorter than the complete sequence, but should be at least 15, preferably at least 25, more preferably at least 40 nucleotides in length. Longer probes may be used. Both DNA and RNA probes can be used. The probe is typically labeled to detect the corresponding gene (eg, with ³² P, ³ H, ³⁵ S, biotin or avidin).

  Hybridization is performed under low to high stringency conditions (ie, 5 × SSPE, 0.3% SDS, 200 μg / ml apical denatured salmon sperm DNA, low, medium and high stringency conditions, respectively) according to standard Southern blotting techniques. Prehybridization and hybridization at 42 ° C. in either%, 35% or 50% formamide), oligo corresponding to the polypeptide coding part of the nucleic acid sequence shown in SEQ ID NO: 1 or contained in pJaL499 Shows that a nucleic acid sequence hybridizes to a nucleotide probe.

  Thus, screening a genomic library, cDNA library or combinatorial chemistry library prepared from another filamentous strain for DNA that hybridizes with the probe and encodes dimethylallyl-cycloacetoacetyl-L-tryptophan synthase Can do. Genomic DNA or other DNA from another filamentous strain can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or isolated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier material. In order to identify clones or DNA homologous to SEQ ID NO: 1, a carrier material is used in Southern blotting, the carrier material using 2 × SSC, 0.2% SDS, preferably at least 50 ° C., more preferably Is at least 55 ° C., more preferably at least 60 ° C., more preferably at least 65 ° C., even more preferably at least 70 ° C., most preferably at least 75 ° C., and a final wash three times for 30 minutes each. Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using X-ray film.

  In a preferred embodiment, the dimethylallyl-cycloacetoacetyl-L-tryptophan synthase of the present invention is an Aspergillus strain, more preferably A. For example, the polypeptide has the amino acid sequence of SEQ ID NO: 2.

  An “isolated” dimethylallyl-cycloacetoacetyl-L-tryptophan synthase, as defined herein, is a polypeptide that is essentially free of other polypeptides, such as at least about 20 as measured by SDS-PAGE. A polypeptide that is% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and most preferably about 95% pure .

  The invention also relates to an isolated nucleic acid sequence encoding dimethylallyl-cycloacetoacetyl-L-tryptophan synthase obtained from a filamentous fungus. In a more preferred embodiment, the nucleic acid sequence is an Aspergillus sp. It is obtained from oryzae and has the nucleic acid sequence set forth in SEQ ID NO: 1. In another preferred embodiment, the nucleic acid sequence is the sequence contained in plasmid pJaL499. The invention also encompasses nucleic acid sequences that differ from SEQ ID NO: 1 due to the degeneracy of the genetic code. The present invention also relates to a partial sequence of SEQ ID NO: 1 or a partial sequence of the polypeptide coding portion of pJaL499, which encodes a polypeptide fragment having dimethylallyl-cycloacetoacetyl-L-tryptophan synthase activity. The partial sequence of SEQ ID NO: 1 or the partial sequence of the polypeptide coding part of pJaL499 is the polypeptide of SEQ ID NO: 1 or pJaL499, except that one or more nucleotides are deleted from the 5 'and / or 3' end A nucleic acid sequence encompassed by a coding portion. Preferably, the subsequence of SEQ ID NO: 1 comprises at least 870 nucleotides, more preferably at least 960 nucleotides, and most preferably at least 1050 nucleotides.

  The nucleic acid sequence can be obtained from a microorganism that is the taxonomic equivalent of Aspergillus oryzae.

  Techniques used to isolate or clone such nucleic acid sequences are described below. As used herein, the term “isolated nucleic acid sequence” refers to a nucleic acid sequence that is essentially free of other nucleic acid sequences, eg, at least about 20% pure, preferably at least as measured by agarose gel electrophoresis. A nucleic acid sequence that is about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure. The nucleic acid sequence can be of genomic DNA, cDNA, RNA, semi-synthetic, synthetic origin or any combination thereof.

  Alteration of the nucleic acid sequence encoding the dimethylallyl-cycloacetoacetyl-L-tryptophan synthase of the invention may be required for the synthesis of substantially the same enzyme as the polypeptide. “Substantially the same” as dimethylallyl-cycloacetoacetyl-L-tryptophan synthase refers to a non-natural form of the enzyme. Their dimethylallyl-cycloacetoacetyl-L-tryptophan synthase may differ from the enzyme isolated from its native source by some method of operation. For example, it may be important to synthesize mutants of the enzyme that differ in specific activity, thermal stability, optimum pH, etc. using site-directed mutagenesis. A similar sequence may be a nucleic acid sequence given as the polypeptide coding portion of SEQ ID NO: 1, for example based on that partial sequence and / or without giving another amino acid sequence of the polypeptide encoded by the nucleic acid sequence and It can be made by introducing nucleotide substitutions corresponding to the codon usage of the host organism for the production of the peptide, or by introducing nucleotide substitutions that give different amino acid sequences. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.

  It will be appreciated by those skilled in the art that such substitutions can be made outside the region critical to the function of the molecule and still result in biologically active dimethylallyl-cycloacetoacetyl-L-tryptophan synthase. It will be obvious. Amino acid residues that are essential for the activity of dimethylallyl-cycloacetoacetyl-L-tryptophan synthase encoded by the isolated nucleic acid sequences of the present invention and therefore preferably not subject to substitution are site-directed mutagenesis or They can be identified according to techniques known in the art such as alanine scanning mutagenesis (see, eg, Cunningham & Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resulting mutant molecule is tested for dimethylallyl-cycloacetoacetyl-L-tryptophan synthase activity, for the activity of the molecule. Identify the amino acid residues that are important. Substrate-enzyme interactions can also be examined by analysis of three-dimensional structures measured by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (eg de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

  Preferred uses for the nucleic acid sequences of the present invention or homologues or fragments thereof are specific host cells, especially A. Removing or reducing dimethylallyl-cycloacetoacetyl-L-tryptophan synthase activity in oryzae cells, thereby reducing or eliminating CPA production from said cells.

  The invention also relates to constructs for expression of the sequences, recombinant expression vectors and host cells containing the nucleic acid sequence of SEQ ID NO: 1 or the polypeptide coding part of pJaL499, partial sequences or homologues thereof. The constructs and vectors can be made as described herein. The host cell can be any cell suitable for expression of the nucleic acid sequence and can be selected, for example, from the parental or mutant cells described herein.

Genetic modification of the host cell In order to significantly reduce the expression level of one or more toxins, the host cell of the invention is genetically modified, which can be achieved using standard techniques known to those skilled in the art. it can. The gene sequence responsible for the production of toxin activity can be inactivated or partially or completely removed. Thus, the Aspergillus mutant host cells of the invention express a reduced or undetectable level of one or more toxins.

  In certain embodiments, inactivation is obtained by alteration of each structural or regulatory region (eg, gene) involved in the formation or secretion of a particular toxin. Known useful techniques include, but are not limited to, specific or random mutagenesis, PCR-generated mutagenesis, site-specific DNA deletion, insertion and / or substitution, gene disruption or gene replacement, antisense technique, or those And the like.

  Mutagenesis can be performed using an appropriate physical or chemical mutagenesis agent. Examples of physical or chemical mutagens that are suitable for the purposes of the present invention include, but are not limited to, UV irradiation, ionizing irradiation, such as gamma irradiation, hydroxylamine, N-methyl-N'-nitro-N- Nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, and nucleotide analogs. When using such agents, the cells to be mutated are typically incubated in the presence of a specific mutagen under appropriate conditions and selected for cells that exhibit significantly reduced target toxin production. By doing so, mutagenesis is performed.

  Reduction or elimination of production of a particular toxin by a host cell can also be achieved by altering nucleotide sequences that are involved in or otherwise required for production or secretion of the toxin. For example, the nucleotide sequence may be a gene product having a necessary function in the pathway leading to toxin production. The nucleotide sequence can be, for example, that shown in SEQ ID NO: 1 or the polypeptide coding portion of pJaL499. Alteration can be accomplished by the introduction, substitution or removal of one or more nucleotides in the nucleotide sequence or in regulatory elements necessary for transcription or translation of the sequence. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, removal of the start codon or alteration of the transcription reading frame into the nucleotide sequence. Alteration or inactivation of the sequence or its regulatory elements can be accomplished by site-directed or random mutagenesis or PCR-generated mutagenesis according to methods well known in the art. Theoretically, the change can be made in vivo, that is, directly to the cell expressing the toxin gene, but at present it is preferred to make the change in a test tube as exemplified below.

  An example of a convenient way to inactivate or reduce the production of a toxin of interest, such as CPA, in a particular filamentous fungal cell is based on gene replacement, gene deletion or gene disruption techniques. For example, in the gene disruption method, a nucleic acid sequence corresponding to a gene of interest or a gene fragment (for example, the DCAT-S gene of the present invention) is mutagenized in vitro to produce a defective nucleic acid sequence, and then the defective nucleic acid sequence is used. Transform parental cells to generate defective genes. Homologous recombination replaces the defective gene sequence with an endogenous gene or gene fragment. It may be desirable for the defective gene or gene fragment to further encode a marker. The marker can be used for selection of transformants in which the nucleic acid sequence has been altered or destroyed.

  Alternatively, gene alteration or inactivation can be performed by established antisense techniques using nucleotide sequences complementary to the nucleic acid sequence of the gene. More specifically, by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene that can be transcribed in the cell and hybridize to the mRNA produced in the cell, the filamentous fungal cell. Can reduce or eliminate the expression of the gene. Thus, under conditions that allow the complementary antisense nucleotide sequence to hybridize to the mRNA, the amount of translated protein is reduced or eliminated.

  Following mutagenesis or other alterations of the toxin pathway genes, mutants are screened for reduced or eliminated toxin production. Specific examples of methods of screening for toxins are given in the examples below. Alternatively, useful screening assays are described in the “Handbook of Toxic Fungal Metabolites” or are available, for example, at institutions that generally test the level of mycotoxins in food.

  Thus, due to genetic alteration, the Aspergillus mutant host cells of the present invention express significantly reduced levels of toxins. In preferred embodiments, the levels of those toxins expressed by the mutant host cells are individually about 50% or more, preferably about 85% or more, more preferably about 90% or more, most preferably about 95% or more, and more Is preferably reduced by 99% or more. In another preferred embodiment, multiple toxins in the mutant host cells of the invention may be reduced in any combination. In yet another preferred embodiment, the product expressed by the host cell consists essentially of at least one toxin of cyclopiazonic acid, kojic acid, 3-nitropropionic acid, emodin, malformin, aflatoxin, ochratoxin and secaronic acid. Not contained in. In particularly preferred embodiments, the product expressed by the host cell is essentially free of at least cyclopiazonic acid, more preferably at least essentially free of cyclopyrazonic acid and kojic acid or aflatoxin, and most preferably at least cyclocyclozonic acid. Contains no piazonic acid, kojic acid and 3-nitropropionic acid.

In a preferred embodiment, the host cell is one or more of NPA, CPA, kojic acid (KA) or maltolidine; preferably at least two of their toxins, such as NPA and CPA; NPA and KA; CPA and KA; or production of NPA, CPA and KA is reduced or eliminated. Oryzae shares. Furthermore, in addition to the removal or reduction of those one or more toxins, the genes of a particular aflatoxin pathway are preferably inactivated such that the resulting mutant strain is unable to produce aflatoxins. A. The aflatoxin gene from A. flavus is well known, so It can be used to identify the corresponding gene in oryzae, which can then be inactivated by methods known in the art.

In another preferred embodiment, the host cell comprises one or more of malformin (eg, malformin A1 or B), ochratoxin (eg, ochratoxin A) and flavioline, preferably at least two of those toxins, eg Malformin and ochratoxin; malformin and flavioline; ochratoxin and flavioline; and malformin, ochratoxin and flavioline production are reduced or eliminated. A. niger or A. niger A stock of A. ficuum .

In another preferred embodiment, the host cell has reduced or eliminated production of secaronic acid (eg, secaronic acid D) or emodin (a precursor of secaronic acid D), preferably both of these toxins. This is a stock of A. aculeatus .

Methods of Producing Polypeptides The method of the present invention significantly reduces the amount of one or more target toxins, while maintaining the intracellular stability of a genetically altered gene encoding a polypeptide of interest. The characteristics of the mutant host cell in terms of maintenance, cell production capacity, and yield of the polypeptide of interest are substantially retained. More particularly, the method of the present invention allows a host cell to be genetically altered within the structural and / or regulatory regions necessary for the production or secretion of one or more toxins of interest, thereby producing said toxins or Secretion is reduced or eliminated.

  Accordingly, another aspect of the present invention is a method for producing a polypeptide or protein (including heterologous polypeptide or protein) in an Aspergillus mutant host cell of the present invention, wherein the A method comprising introducing a nucleic acid sequence encoding a polypeptide, culturing the mutant host cell in a suitable growth medium, and recovering the polypeptide of interest is provided.

  Thus, the mutant host cell of the invention must contain the structure and regulatory gene regions necessary for the expression of the polypeptide of interest. The nature of such structures and regulatory regions is largely determined by the product of interest and the particular Aspergillus host strain. Genetic design of the host cells of the present invention can be accomplished by one of skill in the art using standard recombinant DNA techniques for host cell transformation or transfection (see, eg, Sambrook et al.).

  Preferably, the host cell is modified by well known techniques for the introduction of a suitable cloning vehicle comprising a DNA fragment encoding the desired polypeptide of interest, ie a plasmid or vector. The cloning vehicle may be introduced into the host cell as a self-replicating plasmid or may be integrated into the chromosome. Preferably, the cloning vehicle comprises one or more structural regions operably linked to one or more suitable regulatory regions.

A structural gene is a region of a nucleotide sequence that encodes a polypeptide of interest. Regulatory regions include promoter regions comprising transcriptional and translational regulatory sequences, terminator regions comprising termination signals, and polyadenylation regions. A promoter, i.e. a nucleotide sequence that exhibits transcriptional activity in a particular host cell, is an extracellular or intracellular protein, preferably an enzyme such as amylase, glucoamylase, protease, lipase, cellulase, xylanase, oxidoreductase, pectinase, cutinase or glycolysis It can be derived from a gene encoding a system enzyme. Examples of suitable promoters for directing transcription of nucleic acid constructs in the methods of the present invention include Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger Neutral α-amylase, Aspergillus niger acid stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase Aspergillus oryzae (Aspergillus oryzae) alkaline protease, Aspergillus oryzae (Aspergillus oryzae) triose phosphate isomerase, Aspergillus nidulans (Aspergillus nidulans) acetamide Midaze (amdS), Fusarium oxysporum (Fusarium oxysporum) promoters obtained from the genes encoding trypsin-like protease (U.S. Patent No. 4,288,627), and variants thereof, an abbreviation and hybrid promoters. Particularly preferred promoters are NA2-tpi promoter (a hybrid of a promoter derived from a gene encoding Aspergillus niger neutral α-amylase and a promoter derived from a gene encoding Aspergillus oryzae triose phosphate isomerase), a glucoamylase promoter, and TAKA amylase promoter.

The cloning vehicle may contain a selectable marker. A selectable marker is a gene whose product provides for biocide resistance, virus resistance, heavy metal resistance, auxotrophic mutants for prototrophic strains, and the like. Selectable markers for filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate) Reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfuryl adenyl transferase) and trpC (anthranilate synthase) and equivalents from another species. Preferred for Aspergillus cells are the AmdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopics .

  Furthermore, the selection may be performed by co-transformation, in which case the transformation is performed using a mixture of two vectors and only one vector is selected.

  Techniques used to link the DNA constructs, promoters, terminators and other elements of the invention, respectively, and insert them into a suitable cloning vehicle containing the information necessary for replication are well known to those skilled in the art (eg, See Sambrook et al., 1989, supra).

  Mutant filamentous fungal cells are cultured in a normal medium suitable for production of the polypeptide of interest using methods known in the art. For example, the cells may be small or large in shake flask cultures, laboratory or industrial fermenters performed in appropriate media and under conditions that allow expression and / or isolation of the heterologous polypeptide. It can be cultured by scale fermentation (including continuous fermentation, batch fermentation, fed-batch fermentation or solid phase fermentation). Culturing is performed in a suitable normal medium containing a carbon source, a nitrogen source and inorganic salts using techniques known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions (eg in the catalog of the American Type Culture Collection (ATCC)). The secreted polypeptide can be recovered directly from the medium.

  The polypeptide can be detected using methods known in the art that are unique to the polypeptide. These detection methods include the use of specific antibodies, formation of enzyme products, disappearance of enzyme substrates, or SDS-PAGE. For example, the activity of the polypeptide may be measured using an enzyme assay. Methods for measuring enzyme activity are known for various enzymes.

  The resulting polypeptide can be isolated by methods known in the art. For example, it can be isolated from normal media by routine procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. The isolated polypeptide can then be used, without limitation, chromatography (eg, ion exchange, affinity, hydrophobic, isoelectric point and size exclusion), electrophoresis (eg, preparative isoelectric focusing), fractional solubility ( Further purification can be achieved by various known methods including, for example, ammonium sulfate precipitation or extraction methods (see, eg, Protein Purification, J.-C. Janson & Lars Ryden, VCH Publishers, New York, 1989). about).

Product The desired end product, ie the polypeptide of interest expressed by the Aspergillus mutant host cell, may be any homologous or heterologous protein or peptide.

  The polypeptide can be any polypeptide that is heterologous to the mutant filamentous fungal cell. The term “polypeptide” does not refer to a specific length of the encoded product, and thus encompasses peptides, oligopeptides and proteins. A heterologous polypeptide may be a modified variant of a polypeptide. The term “heterologous polypeptide” is defined herein as a polypeptide that is not native to a filamentous fungal cell. The mutant filamentous fungal cell may contain one or more copies of the nucleic acid sequence encoding the heterologous polypeptide.

  In the method of the present invention, mutant filamentous fungal cells may be used for recombinant production of polypeptides native to the cells. For example, to enhance the expression of the polypeptide, to facilitate the transport of the native polypeptide of interest to the outside of the cell through the use of a signal sequence, and for the gene encoding the polypeptide normally produced by the cell In order to increase the copy number, the native polypeptide can be produced recombinantly by placing the gene encoding the polypeptide under the control of another promoter. As long as the present invention involves the use of genetic elements that have been engineered to function in such a way that such expression does not normally occur in the host cell, or involves genetic elements that are not native to the cell, Such recombinant production is also encompassed within the term “heterologous polypeptide”.

  In a more specific embodiment, the product is a therapeutically effective peptide or protein, such as hormones, in particular insulin, growth hormone, glucagon or somatostatin; interleukins, in particular interferons; hematopoietic growth factors, in particular PDGE (platelet derived growth factor), EPO (erythropoietin) or TPO (thrombopoietin); proteases, in particular factor VII, factor VIII, urokinase, chymosin or TPA; or serum albumin.

  In another preferred embodiment, the product is an enzyme of fungal or bacterial origin. The enzyme is preferably a glycosidase enzyme, such as an amylase, in particular α-amylase, β-amylase or glucoamylase; glucan 1,4-α-glucosidase; aminopeptidase; carbohydrase; carboxypeptidase; catalase; Β-glucanase or endo-1,3 (4) -β-glucanase; cellulose-1,4-β-cellobiosidase; chitinase; cutinase; cyclodextrin glycosyltransferase; deoxyribonuclease; galactanase; galactosidase, especially α-galactosidase β-galactosidase; endoglucanase, in particular endo-1,3-β-glucanase, endo-1,3-α-glucanase, endo-1,2-β-glucanase or Endo-1,6-β-glucanase; glucosidase; especially α-glucosidase or β-glucosidase; invertase; laccase; lipolytic enzyme, especially lipase, esterase, phospholipase or lyso-phospholipase; lyase or pectate lyase; Mugalase; oxidase or oxidoreductase, such as peroxidase or polyphenol oxidase; oxygenase; pectinase, endopeptidase or exopeptidase; phytase; polygalacturonase; protease; ribonuclease; transglutaminase; 4-β-xylanase or xylan-endo-1,3-β-xylosider It is.

  In another preferred embodiment, the product is a hybrid polypeptide, such as prochymosin and protrypsin-like protease. The heterologous polypeptide expressed by the host cell is obtained as a precursor protein such as a zymogen, a hybrid protein, a pro-sequence or a pre-pro sequence under appropriate conditions, eg, in the absence of substantial protease activity. Or any other immature form.

  The invention will be further described with reference to the following examples, which should not be construed as limiting the scope of the invention as defined in the claims.

Aspergillus mutants in which the silent toxin gene has been removed The biosynthetic pathway of aflatoxins has been studied in the aflatoxin-producing species Aspergillus flavus and Aspergillus parasites. Both species have been shown to have numerous genes identified and mapped as large clusters [Woloshuk, CP & Prieto, R., FEMS Microbiology Letter (1998) 160: 169-176]. Several genes have been cloned and sequenced, such as aflR, which encodes an expression regulator gene for alternative pathway genes, and omtA, which encodes O-methyltransferase.

  The aflatoxin gene is A. Although present in the genome of Oryzae, it is not expressed from this species. Even if no aflatoxins are expressed, it is advantageous to remove one or more of their silent aflatoxin pathway genes. Aspergillus mutants in which the aflatoxin gene, eg aflR and / or omtA gene has been removed, eg A. Orize mutants are advantageous because it is not necessary to test new mutant strains for the production of one or more aflatoxins in question.

  Thus, in one aspect, the invention relates to Aspergillus mutant cells suitable for the expression of heterologous polypeptides from which one or more silent toxin genes have been removed.

  A silent toxin gene has been “removed” in such a way that the silent gene is no longer contained in the mutant cell, for example, gene replacement techniques or gene disruption techniques well known in the art (eg Miller et al., 1985). , Molecular and Cellular Biology, p. 1714-1721) means that the gene (s) in question has been altered or deleted.

  The term “silent” toxin gene means that the toxin is not expressed.

  The toxin gene may be removed by irreversible deletion or destruction of all or part of the toxin gene.

  The term “irreversible deletion or disruption of all or part of a toxin gene” means that the gene does not encode a toxin and naturally back mutates to a gene encoding the toxin (eg during production). It means that the toxin gene in question has been removed or altered in such a way that it cannot be done.

  The Aspergillus cells in question can be any of the above, and the Aspergillus subgroups Eurotium, Ketosartoria, Sclerocreista, Satoia, Neosartorya, Hemicarpenteles (Hemicarpenteles), Petromyces, Emericella, and Fenellia.

  The toxin gene in question encoding one or more toxins includes a toxin selected from the group consisting of cyclopiazonic acid, kojic acid, 3-nitropropionic acid, emodin, malformin, aflatoxin, ochratoxin and secaronic acid.

  Particularly expected are toxin genes encoding aflatoxins, particularly A. It is a toxin gene from the aflatoxin cluster of oryzae, in particular a toxin gene selected from omtA, aflR, pksA, Nor-1, fas-beta, fas-alpha, vber-1, avnA, ord-2.

  Parental Aspergillus cells are Oryzae cells, especially A. It can be oryzae A1560 (IFO 0417).

Experiment
Materials and Methods Strain Aspergillus oryzae (Aspergillus oryzae) A1560 is the same as the IFO 04177 (see below).
Aspergillus oryzae IFO 4177: Available from Institute for Fermentation, Osaka (Fermentation Research Institute; 2-17-25 Juzahoncho, Yodogawa-ku, Osaka). See WO 98/12300.
JaL228: Aspergillus oryzae strain in which the neutral metalloprotease NpI gene is disrupted; the production of this strain is described in WO 98/12300.

BECh 1: The production of this CPA negative Aspergillus oryzae strain is described in Example 1.
BECh 2: The production of this CPA negative KA negative Aspergillus oryzae strain is described in Example 1.
BECh 3: The production of this CPA negative KA negative Aspergillus oryzae strain is described in Example 1.

BZ14: Aspergillus oryzae strain co-transformed with ToC90 and phD450 as described in WO 92/17573.
JaL 250: The production of this Aspergillus oryzae strain is described in Example 6.

Deposit:
E. plasmid containing plasmid pJaL499 The coli strain was deposited with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; Mascheroder Weg 1b, D-38124 Brauschweig) on January 13, 1999, and was given the deposit number DSM 12622.

2. gene
DMAT-S: This gene encodes dimethylallyl-L-tryptophan synthase, an enzyme involved in ergot alkaloid biosynthesis.
DCAT-S: This gene encodes dimethylallyl-cycloacetoacetyl-L-tryptophan synthase, an enzyme involved in cyclopiazonic acid (CPA) biosynthesis.
pyrG: This gene encodes orotidine-5'-phosphate decarboxylase, an enzyme involved in uridine biosynthesis.

3. Plasmid
pAHL: This plasmid is described in WO 97/07202.
pCaHj483: This plasmid is described in WO 98/00529.
pCaHj493: This plasmid is described in Example 2.
pJaL335: This plasmid is described in WO 98/12300.
pJaL499: This plasmid is described in Example 4.

4). Media and solutions The reagents used as buffers and substrates were of at least reagent quality.
Screening medium 1 (per liter)
Mannitol 30 g
Glucose 10 g
Succinic acid 10 g
Casamino acid 3 g
KH ₂ PO ₄ 1 g
MgSO ₄・ 7H ₂ O 0.3 g
FeSO ₄・ 7H ₂ O 0.2 g
2,6-dichloro-4-aniline 2 ppm
Agar 20 g
Adjust to final pH 5.6 with 14% NH ₄ OH.

Cove N
Cove salt solution 50 ml
Sorbitol 218 g
Dextrose 10 g
Potassium nitrate 2.02 g
Agar 35 g
1000 ml deionized water

Cove salt solution (per liter)
KCl 26 g
MgSO ₄ 26 g
KH ₂ PO ₄ 76 g
Trace metal solution 50 ml
CHCl ₃ 2 ml

Trace metal solution (per liter)
Na ₂ B ₄ O ₇・ 10H ₂ O 40 mg
CuSO ₄・ 5H ₂ O 400 mg
FeSO ₄・ 7H ₂ O 800 mg
MnSO ₄・ 2H ₂ O 800 mg
Na ₂ MoO ₄・ 2H ₂ O 800 mg
ZnSO ₄・ 7H ₂ O 8000 mg

G1-gly
Yeast extract 18 g
Glycerol 87% 24 ml
Pluronic PE6100 1 ml
To 1000 ml with tap water

1/5 MDU-2BP
Maltose 9 g
MgSO ₄・ 7H ₂ O 0.2 g
NaCl 0.2 g
K ₂ SO ₄ 0.4 g
KH ₂ PO ₄ 2.4 g
Yeast extract 1.4 g
AMG trace metal 0.1 ml
Pluronic PE6100 0.02 ml
Final pH 5.0 in 1000 ml deionized water; add 1.0 ml 50% urea before inoculation.

MDU-IB (per liter)
Maltodextrin MD01 45.0 g
MgSO ₄・ 7H ₂ O 1.0 g
NaCl 1.0 g
K ₂ SO ₄ 2.0 g
KH ₂ PO ₄ 12.0 g
Yeast extract 7.0 g
AMG trace metal 0.5 ml
Pluronic PE6100 1 ml
Final pH 5.0; 1.3 ml of 50% urea is added per 100 ml medium before inoculation.

AMG trace metal solution (per liter)
FeSO ₄・ 7H ₂ O 13.9 g
MnSO ₄・ H ₂ O 8.45 g
ZnCl ₂ 6.8 g
CuSO ₄・ 5H ₂ O 2.5 g
NiCl ₂・ 6H ₂ O 2.5 g
Citric acid ≥3.0 g
Trace metal solution 1 ml

KM2 medium (per liter)
Yeast extract 2.5 g
KH ₂ PO ₄ 10 g
MgSO ₄・ 7H ₂ O 0.5 g
KCl 0.5 g
FeSO ₄ 0.01 g
Glucose 100 g
Adjust final pH to 6.0 KMZ medium is coagulated with 20 g / l agar before use on solid plates.
Add 300 μl / l Triton X-100 as a colony growth limiting agent.

Nakamura medium (per liter)
Sucrose 50 g
Peptone 20 g
KH ₂ PO ₄ 5 g
CaHPO ₄ 2.5 g
MgSO ₄ 2.5 g
Adjust final pH to 6.0.

5. Assay A. Assay method for CPA by HP capillary electrophoresis
Capillary electrophoresis by solid-phase extraction on Supelclean LC-18 SPE tubes (Supelco pre-packed 3 ml columns from catalog No. 5-7012 conditioned with 2 ml methanol and 2 ml Milli-Q water) ( Prepare a 1 ml aliquot of the sample for CE). Use a suction manifold to force the solution through the column. After washing with 3 ml of Milli-Q water, the sample is eluted with 3 ml of methanol. The eluate is subjected to CE analysis without further workup. Optionally formed precipitate is removed by centrifugation.

  CE analysis is performed using a Hewlet-Packard photodiode array CE device (3D-CE). The sample is injected over 10 seconds under a hydrostatic pressure of 3400 Pa (34 mbar). Using a 50 mm capillary (effective length 56 cm) at 30 ° C., it is conditioned for 1 minute with 0.1 M NaOH and then with 100 mM boric acid / NaOH pH 9.1 for 5 minutes. Set the voltage to 17 kV. A UV spectrum is always collected to identify the peak, but the migration is followed at 280 nm. Commercially available cyclopiazonic acid is used as a standard sample (Sigma Co., St. Louis MO, USA, Catalog No. C1530, minimum 98% purity). The lower limit of sensitivity of this method is about 1 ppm.

B. Assay Method for CPA by Thin Layer Chromatography (TLC) Analysis of plate cultures Agar plugs were prepared as described in Filtenborg O., Frisvad JC & Svendsen JA: "Simple Screening Method for Mold Producing Intracellular Mycotoxins in Pure Cultures", Applied and Environmental Microbiology (1983) 45: 581-585. analyse.

2. A 10 μl sample of the liquid culture assay supernatant is applied to the diagonal edges of a TLC plate (Merck Silica Gel 60). Cyclopiazonic acid (Sigma C 1530) dissolved and diluted to 50 ppm, 25 ppm, 5 ppm and 2.5 ppm in a methanol: chloroform (1: 2 by volume) mixture is used as a standard. The plate is first developed in CAP (chloroform: acetone: propan-2-ol = 85: 15: 20 by volume) for 15 minutes, dried, then inverted upside down and the other half is TEF (toluene: ethyl acetate: Develop for 15 minutes in formic acid = volume 5: 4: 1).

Alternatively, the plates are developed as above in EMA (ethyl acetate: methanol: 25% ammonium hydroxide = 16: 8: 2 by volume) and TEF for 15 minutes each.
Plates are completely dried (1 hour) in a fume hood and then sprayed with Ehrlich reagent (2 g 4-dimethylaminobenzaldehyde dissolved in 85 ml 96% ethanol plus 37% hydrochloric acid). To do.

  CPA has a typical low mobility in the CAP system (neutral system) and is observed as a bluish-purple mushroom-type spot, whereas in the acidic TEF system, the application position and the front line of the developing agent are observed. This produces a typical smear that is elongated in the middle. In the EMA system (basic / alkaline system), cyclopiazonic acid is concentrated in small dense spots.

According to direct visual inspection of the plate, a CPA concentration of ≧ 2.5 ppm can be observed as a purple band smear or spot (depending on the development system). By scanning the TLC plate on a tabletop flatbed scanner and then processing and enhancing the electronic image using an appropriate image processing program (Paint Shop Pro 4 in this case), the sensitivity is increased 5 to 10 times. .
The overall sensitivity of this analysis is about 0.5-1 ppm CPA (without extraction); using extraction increases sensitivity at least 10-fold.

C. Assay method for kojic acid by capillary electrophoresis Supelclean LC-18 SPE tube conditioned with methanol and 10 mM boric acid / NaOH, 4 M KCl, pH 9.1 (Supelco prepacked 3 ml column from catalog No. 5-7012) Prepare a 1-3 ml aliquot of the sample for CE analysis by solid phase extraction above. Use a suction manifold to force the solution through the column. After washing with 3 ml 10 mM boric acid / NaOH, 4 M KCl pH 9.1 and 0.3 ml 10 mM boric acid / NaOH pH 9.1, elute the sample with 7.5 ml 10 mM boric acid / NaOH pH 9.1 . Without further workup, the eluate is subjected to CE analysis according to the procedure described above for cyclopiazonic acid. Optionally formed precipitate is removed by centrifugation. The lower limit of sensitivity of this method is about 6 ppm.

D. Assay Method for Kojic Acid by Thin Layer Chromatography An aliquot of the sample is applied to the diagonal end of the TLC plate as described above for CPA and developed using the same solvent system as CPA. Spray dried plates with 1% FeCl ₃ in 0.1 M HCl. The presence of kojic acid in the sample is indicated by a red spot and compared to the intensity of the red spot produced by pure kojic acid applied as a control. The lower limit of detection is 50 ppm.

E. Assay Method for 3-NPA by Capillary Electrophoresis The need for sample purification in preparation for capillary electrophoresis (CE) analysis depends on the conductivity of the sample. If the conductivity is less than 10 mS, the sample is purified by ion exchange on a Varian SAX anion exchanger (Varian Instruments, Palo Alto CA) using 0.1 M KCl as the elution buffer. If the conductivity is greater than 100 mS, use 2-butanol to extract the sample. In that case, after precipitation formation by acidification / high salt treatment and redissolution of the precipitate in 10 mM Tris / HCl pH 7.0, a 2 ml sample is extracted with 6 ml butanol.

  HP-CE instrument diode array detection is used using a capillary of uncoated silica with a diameter of 50 μm and an effective length of 56 cm using a conditioning buffer of 25 mM boric acid / phosphate pH 7.6 at a temperature of 30 ° C. Samples are injected under hydrostatic pressure over 20 seconds. Set the voltage to 30 kV. The lower limit of detection of this method is 6 ppm.

F. Assay Method for 3-NPA by Thin Layer Chromatography Fermentate spots are applied and developed on TLC plates as described for CPA. They were then subjected to diazotized p-nitroaniline as described by W. Majak & RJ Bose, “Chromatographic methods for the isolation of miserotoxin and the detection of aliphatic nitro compounds”, Phytochemistry (1974) 13: 1005-1010. Spray. The intensity and position of the spot relative to the control substance is a measure of the 3-NPA concentration. The detection level on the TLC plate is 25-50 ppm.

  Alternatively, 3-NPA is analyzed spectrophotometrically (λ = 540 nm) by adding 50 μl 1 M NaOH and 70 μl diazotized p-nitroaniline to a 100 μl sample. The detection level is 5-10 ppm.

Example 1
A. A. Preparation of CPA-negative strain derived from Oryzae BZ 14 Lyophilized spores of Aspergillus oryzae BZ14 were irradiated with gamma rays at an optimal dose of 1000 Gy to 1250 Gy, and then the spores at a density of 25-50 colonies / 9 cm plate Inoculated on screening medium 1. A colony producing cyclopiazonic acid forms a red back surface (under the colony) on the screening medium 1 to form a red insoluble CPA-Fe complex.

  Approximately 50,000 colonies were screened from irradiated spores and 154 CPA-deleted colonies characterized by a milky white (cream / white) appearance were isolated. After re-separation, 64 strains retained the non-red back side on the screening medium 1 plate. No TPA was detected in 52 strains by TLC plug assay. The strains were then cultured in MDU-1B medium under toxin-induced (34 ° C., 250 rpm, 5 days) shake flask fermentation conditions. 36 strains did not show detectable levels of CPA in the supernatant as measured by TLC.

B. A. Preparation of CPA negative strain BECh 1 derived from Oryzae JaL228
JaL228 lyophilized spores were gamma irradiated and screened as above. Putative CPA deletion isolates were cultured in shake flasks on MDU-1B medium under toxin-inducing conditions (34 ° C., 250 rpm for 5 days), and supernatants were analyzed for CPA by the TLC method. The supernatant was tested as is or as an extract.

  For extraction, 50 ml of all samples were acidified with 10 ml of 0.1 M HCl. The mixture was then shaken vigorously with 70 ml methanol / chloroform (1: 2) for 3-5 minutes. After phase separation (after about 3 hours), the lower layer (about 25 ml) was transferred to a 300 ml beaker and the chloroform was evaporated. The residue was redissolved in 5 ml of chloroform, transferred to a 25 ml beaker and the chloroform was evaporated. The residue was dissolved in 100 μl chloroform.

10 μl of supernatant or chloroform extract was applied to the diagonal edges of a 20 cm × 20 cm TLC plate and processed as described in the previous section on the assay.
Three strains (isolates) containing BECh 1 did not produce CPA.

C. Generation of CPA negative KA negative strains BECh 2 and BECh 3
BECh 1 was cultured on the Cove N slope. Spores were suspended in 0.01% Tween to a density of 3-5 × 10 ⁶ and subjected to short wave UV irradiation (254 nm from germicidal lamp). Spores irradiated with UV doses that yielded 1-5% viability were used for subsequent screening.

Irradiated spores were diluted in KM2 medium to a density of about 0.7 spores / 100 μl and 100 μl was inoculated into each well of a 96 well microtiter plate. Cultures were placed in a humid chamber and incubated at 34 ° C for 5-7 days in a quiescent state. Then 40 μl of 1% FeCl ₃ /0.1 M HCl was added to each well with signs of growth.
Dark red color indicates kojic acid (KA) production; no color development indicates colonies lacking kojic acid production.

Alternatively, spores were seeded on coagulated KM2 medium (growth restricted with Triton X-100) and when mature colonies were observed, the plates were irrigated with 1% FeCl ₃ in 0.1 M HCl. Those without a red area around the colony indicate a putative non-kojic acid producing strain.

From about 7000 microtiter cultures, 132 putative KA negative colonies, ie those that did not develop a color reaction with FeCl ₃ , were isolated. However, when tested on primary KM2 screening agar plates, none of the colonies were confirmed to be KA negative.

  Retesting of negative colonies under KA-induced conditions using both medium on solid medium and in stationary liquid KM2 medium (30 °) narrowed the number of KA negative mutants to 11. When these strains were tested in shake flasks, 8 strains produced KA. The remaining three did not give a color reaction either when examined by TLC or when the supernatant was assayed directly. As expected, KA was produced by the control strain BECh 1 when grown in similar parallel cultures. One of the isolates showed an abnormal morphology. After prolonged growth on MDU-1B, the remaining two isolates were tested for both CPA and KA. Neither CPA nor KA was detected. The two strains were named BECh 2 and BECh 3.

D. 3-NPA negative A. already CPA negative and KA negative. Preparation of oryzae strain
Each of the BECh2 and BECh3 strains was subjected to UV mutagenesis as described above. Irradiated spores were diluted in 96-well microtiter plates to a density of 0.7 viable spores / 100 μl Nakamura medium. Incubate for 5-7 days at 30 ° C. in a humid chamber.

  Fermentation broth samples from wells showing growth were transferred to new microwell plates and analyzed spectrophotometrically or applied to TLC plates. Strains that were 3-NPA negative were re-cultured in shake flasks with Nakamura medium and analyzed for 3-NPA. Strains that were 3-NPA negative were transformed with pCaHj 493 as described in Example 2 and the transformants were treated as described in Example 3.

Example 2
A. Expression of lipase gene in oryzae strains JAL228 and BECh 1 Construction of plasmid pCaHj493 The lipase plasmid pAHL (WO 97/07202) was digested with BamHI and SalI, and the 916 bp fragment encoding the resulting lipase was isolated.
PCaHj 483 described in WO 98/00529 was digested with BamHI and XhoI, and the 6757 bp vector fragment was ligated with the lipase fragment. The ligation mixture was used to E. coli DH5α cells were transformed and transformants containing the desired plasmid were isolated. The resulting plasmid was named pCaHj493.

B. Transformation of pCaHj 493 into JaL228 and BECh-1 Aspergillus oryzae strains JaL228 and BECh1 were transformed with pCaHj493 using selection on acetamide as described in European Patent Application No. 0531372. Transformants were re-separated twice. Spores from the second re-separation of each transformant were tested for lipase production in shake flasks and in microtiter dish cultures.

Example 3
A. CPA negative A. Oryzae strain and CPA positive Lipase production in oryzae strains 18 JaL228 transformants and 30 BECh 1 transformants prepared as described in Example 2 were examined for lipase production in shake flask culture.

  100 ml G1-Gly medium in which Cove N slant cultures of transformants were obtained using 10 ml 0.1% Tween solution and the spore suspension was placed in a 500 ml two-baffled shake flask It was used as an inoculum. The culture was incubated for 24 hours at 250 rpm on a rotary shaker at 34 ° C. 10 ml of the G1-Gly culture was then transferred to 100 ml 1/5 MDU-2BP in a 500 ml shake flask and further incubated at 34 ° C. at 250 rpm.

  Samples were taken after 50 hours, filtered through Miracloth and centrifuged (4000 × g). Simple radial immunodiffusion (Scand, J. Immunol. Vol. 17, suppl. 10, 41-56, (1983), "Handbook of Immunoprecipitation-in-Gel Techniques", NH Axelsen, Blackwell Scientific Publications, 1983) Used to detect the lipase concentration (expressed in LU / ml) in the supernatant.

  Thirty CPA negative BECh 1 transformants had the same or higher lipase yield as 18 CPA positive JaL228 transformants. Table 2 gives an overview of the distribution.

B. CPA negative A. Oryzae strain and CPA positive Xylanase production by oryzae strains Ten strains that did not show detectable CPA production (prepared in Example 1A) and three strains that could detect CPA were evaluated for xylanase production. The results are summarized in Table 1 below. The column in the second column is a simple radial immunodiffusion method [Scand, J. Immunol. Vol. 17, suppl. 10, 41-56 (1983), "Handbook of Immunoprecipitation-in-Gel Techniques", edited by NH Axelsen, Blackwell. Scientific Publications, 1983] shows the amount of xylanase produced in shake flask culture measured in fungal xylanase units (FXU).

  This result indicates that the CPA negative strain can produce xylanase in an amount comparable to the CPA positive strain.

C. Lipase production in CPA negative KA negative strains Two CPA negative KA negative strains, BECh 2 and BECh 3, were transformed with plasmid pCaHj 493 as described in Example 2. The obtained transformant was separated into spores twice. Spores from the second re-separation of each transformant were tested for lipase production as described in Example 2.

  Table 2 shows the frequency distribution of lipase yields of transformants from these two strains. The results are as follows. Compared to the values given for the oryzae strains BECh 1 and JaL228, it is shown that there is no damage to the expression capacity obtained.

  MDU1B shake flasks for CPA production were inoculated from the same spore suspension used for the lipase production culture and incubated for 5 days as described above. DPA analysis was performed according to item 5B2. None of the BECh 1 strains produced CPA, while 17 of the 18 JaL228 strains produced more than 25 ppm CPA and most produced more than 100 ppm CPA.

Example 4
A. Identification and genomic cloning of the Oryzae DCAT-S gene A. Identification of oryzae dimethylallyl-cycloacetoacetyl-L-tryptophan synthase (DCAT-S) gene The cDNA clone (pJaL499) contains the DNA sequence shown in SEQ ID NO: 1. It has been identified to be related to CPA biosynthesis due to its homology to the dimethylallyl tryptophan synthase (DMAT-S) gene of Claviceps purpurea . A. Sequencing of the oryzae cDNA clone encodes a 473 amino acid polypeptide (SEQ ID NO: 2) that is 1393 base pairs in length (SEQ ID NO: 1) and 42.1% identical to DMAT-S from Krabiceptus purpurea showed that.

A. Orize DCAT-S polypeptide is involved in β-CPA biosynthesis from cycloacetoacetyl-L-tryptophan (Nethling DC & McGrath, Can. J. Microbiol. (1977) 23: 856-872).
Chromosomal DNA was prepared from JaL228 strain and BECh 1 strain. DNA was digested with BglII, NcoI, XhoI and SpeI and analyzed by Southern blotting using a 1 kb ³² P-labeled BglII DNA fragment from pJaL499 containing the DCAT-S gene as a probe. Southern blot analysis showed that CPA production strain JaL228 has one DCAT-S gene, while BECh 1 has its DCAT-S gene deleted from the chromosome.

B. Cloning of genomic clones of DCAT-S gene Using the following restriction enzymes: EcoRI, SalI, BbuI, XhoI and XbaI, using a 1 kb ³² P-labeled BglII DNA fragment from pJaL499 containing the DCAT-S gene as a probe, A . Genome restriction mapping of the Oryzae DCAT-S gene was performed (Fig. 1). This indicates that there is only one copy of the DCAT-S gene.

  JaL228 genomic DNA was partially digested with Tsp509I or run on a 0.7% agarose gel. A fragment having a size between 7 kb and 10 kb was purified.

Then cloned into Lambda ZAP II using the supplied protocol Purification already DNA to the manufacturer (Stratagene ^R). In accordance with the instructions given by the manufacturer, any cloned insert contained within the λ vector was excised in vivo and recirculated to produce a fazimide containing the cloned insert for the DNA library. It is outlined in standard method reference books (eg J. Sambrook, EF Fritsch & T. Maniatis (1989) "Molecular Cloning: A Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). As described above, clones encoding the DCAT-S gene were screened by colony hybridization using a 1 kb ³² P-labeled DNA BglII fragment from pJaL499 containing the DCAT-S gene as a probe.

Example 5
Production of Aspergillus oryzae CPA negative strain by gene disruption of dimethylallyl-cycloacetoacetyl-L-tryptophan synthase (DCAT-S) of Aspergillus oryzae Using the oryzae pyrG gene, A. DCAT-S gene in pyrG-negative strain of oryzae was replaced by one-step gene replacement method [BL Miller et al., Mol. Cell. Biol. (1985) 5: 1714-1721; G. May, Applied Molecular Genetics of Filamentous Fungi, 1- 25, JR Kinghorn & G. Turner, Blakie Academic and Professional, 1992].

A. Construction of DCAT-S disrupted plasmid Plasmid pJaL499 is digested with SacII, treated with Klenow polymerase to make blunt ends, and treated with bacterial alkaline phosphatase according to the manufacturer's instructions (Boehringer Mannheim) to remove the 5 'phosphate group. Removed, then phenol extracted and precipitated.

  The plasmid pJaL335 described in WO 98/12300 was digested with HindIII and A 3.5 kb fragment containing the oryzae pyrG gene was obtained, which was treated with Klenow polymerase to make blunt ends, isolated by gel electrophoresis, and purified. The two pieces were then mixed together and ligated. E. After transformation into E. coli, colonies carrying the correct plasmid were identified by restriction enzyme digestion of the miniplasmid preparation. The construction of the disrupted plasmid (pDCAT-S-pyrG) is summarized in FIG.

B. pyrG negative A. Isolation of oryzae strain JaL250 The oryzae strain JaL228 was screened for resistance to 5-fluoroorotic acid and the natural pyrG mutant was identified. One strain designated JaL250 was identified as being pyrG negative. Since this mutant is uridine dependent, it can be transformed with the wild type pyrG gene and the transformant can be selected by its ability to grow in the absence of uridine.

C. Construction of Aspergillus oryzae DCAT-S deletion strain The 4.9 kb NotI-EcoRI fragment of plasmid pDCAT-S-pyrG was gel purified and purified as described by Christensen et al., Biotechnology (1988) 6: 1419-1422. Used to transform the Oryzae strain JaL250. Transformants were then selected by their ability to grow in the absence of uridine. After re-separating twice, transformants were screened for the ability to produce CPA as described in Example 1.

In order to confirm that the DCAT-S gene was disrupted, chromosomal DNA was prepared from a transformant that did not produce CPA. The DNA was digested with EcoRI and analyzed by Southern blotting using a 1 kb ³² P-labeled DNA BglII fragment from pJaL499 containing the DCAT-S gene as a probe. Transformants with a DCAT-S gene disruption are identified by the migration of the wild-type EcoRI band on 6.3 kb towards the EcoRI band on 9.8 kb.

Example 6
Confirmation that BECh1 and BECh2 lack two genes from the aflatoxin biosynthetic pathway cluster. The presence of aflR and omtA aflatoxin genes from the aflatoxin biosynthetic pathway cluster was explored in oryzae IFO 4177 and its many derivatives. By PCR using primers 5956 (5′-GGATCCAGGGCTCCCTGGAG-3 ′) (SEQ ID NO: 3) and 5955 (5′-CCTGACCAGCCAGATCTCCT-3 ′) (SEQ ID NO: 4), A. The aflR homologue was isolated from the genomic DNA of oryzae IFO4177. A 0.9 kb PCR fragment was obtained and cloned into the vector pCR2 obtained from Invitrogen. The identity of the cloned fragment was confirmed by sequence analysis of the resulting plasmid pToC280 using M13 forward (-40) primer and reverse primer. In addition, the omtA homologue was isolated from genomic IFO4177 DNA by PCR using primers 6120 (5'-AGTGAGAGAACTCCCTCCTC-3 ') (SEQ ID NO: 5) and 6121 (5'-CCATATCTTCTCAGTCTCCA-3') (SEQ ID NO: 6). did. Confirm the identity of the cloned fragment by obtaining a 1.2 kb fragment, cloning it into the vector pCR2 from Invirtogen, and sequencing the resulting plasmid pToC276 using M13 forward (-40) and reverse primers did.

The above aflR and omtA cloned fragments were used as ³² P-labeled probes in hybridization experiments. Genomic DNA from IFO4177, JaL228, BECh1 and BECh2 was digested with the restriction enzyme EcoRI and the resulting fragments were separated on a 0.7% agarose gel. DNA was blotted onto the membrane and hybridized under stringent conditions using the two ³² P-labeled probes one by one [Method: Sambrook, EF Fritsch, T. Maniatis (1989) "Molecular Cloning: A Laboratory Manual ", 2nd edition, Cold Spring Harbor, New York]. The blot showed a positive hybridization signal when both probes from IFO4177 and JaL228 were used, while no band was detected in the lane containing BECh1 and BECh2 DNA. In the IFO4177 and JaL228 lanes, one 3.8 kb fragment could be observed using the omtA probe, and two bands of approximately 0.5 kb and 4.3 kb could be observed using the aflR probe.

  Therefore, A. Oryze IFO4177 contains at least two genes from the aflatoxin biosynthetic pathway, namely the aflR gene and the omtA gene. A. Flaves and A.M. In Parasitics, the two genes are separated by approximately 32 kb [Woloshuk, C.P. & Prieto, R., FEMS Microbiology Letter (1998) 160: 169-176]. Neither of these genes is present in ICh4177 derivatives BECh1 and BECh2.

  The present invention is not to be limited in scope by the specific embodiments disclosed herein, which are provided as illustrations of some aspects of the invention. Any equivalent embodiment can also be included within the scope of the present invention. Indeed, various modifications of the invention will become apparent to those skilled in the art from the foregoing description, in addition to those illustrated and described above. Such modifications are intended to fall within the scope of the claims.

  Various references are cited herein, the entire contents of which are incorporated by reference.

Claims (14)

A parental host cell Aspergillus mutant cell useful for production of the polypeptide of interest, wherein the cell is genetically modified by genomic inactivation of a gene encoding dimethylallyl-cycloacetoacetyl-L-tryptophan synthase Whereby the host cell produces reduced or undetectable levels of cyclopiazonic acid compared to parental Aspergillus cells cultured under the same conditions ,
here,
The gene encoding dimethylallyl-cycloacetoacetyl-L-tryptophan synthase hybridizes with a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 according to standard Southern blotting procedures under high stringency conditions;
The gene encoding dimethylallyl-cycloacetoacetyl-L-tryptophan synthase comprises the nucleotide sequence of SEQ ID NO: 1, or
A cell wherein the dimethylallyl-cycloacetoacetyl-L-tryptophan synthase comprises the amino acid sequence of SEQ ID NO: 2 .   The Aspergillus cell is composed of Aspergillus subgroup Eurotium, Ketosartoria, Sclerocreista, Satoia, Neosartorya, Hemicarpenteles, Petromyces, The cell according to claim 1, which is selected from the group consisting of Emericella and Fenellia.   The Aspergillus cell is A. A. oryzae, A. oryzae A. aculeatus, A. A. nidulans, A. A. ficuum, A. ficuum A. flavus, A. flavus A. foetidus, A. A. soja, A. soja A. sake, A. sake Niger, A. niger A. japonicus, A. japonicus Parasitics (A. parasiticus), and A. The cell according to claim 2, which is selected from the group consisting of A. phoeicus. The cell according to any one of claims 1 to 3 , wherein the polypeptide of interest originates from an Aspergillus host cell. The cell according to any one of claims 1 to 3 , wherein the polypeptide of interest is heterologous to an Aspergillus host cell. The polypeptide of interest is selected from the group consisting of hormones or precursor forms thereof, enzymes or enzyme variants or precursor forms thereof, antibodies or functional fragments thereof, receptors or functional fragments thereof, and reporters. The cell according to any one of to 5 . The enzyme is aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glucosyltransferase, deoxyribonuclease, esterase, galactanase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, From β-glucosidase, invertase, laccase, lipase, lyase, pectate lyase, mannase, mannosidase, mutanase, oxidase, oxygenase, pectinase, endopeptidase, exopeptidase, peroxidase, phytase, polyphenol oxidase, protease, ribonuclease, transglutaminase and xylanase Na Selected from the group, the cell of claim 6. The genome inactivation may include dimethylallyl by specific or random mutagenesis, PCR-generated mutagenesis, site-specific DNA deletion, insertion and / or substitution, gene disruption or gene replacement, antisense technology, or a combination thereof. The cell according to any one of claims 1 to 7 , obtained by modification of a gene encoding cycloacetoacetyl-L-tryptophan synthase. The genome inactivation, dimethylallyl - a gene encoding a cycloalkyl acetoacetyl -L- tryptophan synthase obtained by partially or completely removed from the cell, according to any one of claims 1-7 Cells. At least one toxin selected from the group consisting of kojic acid, 3-nitropropionic acid, emodin, malformin, ochratoxin, aflatoxin, and secaronic acid, which is less in number than the parent Aspergillus cells cultured under the same conditions. The cell according to any one of claims 1 to 9 , which has been genetically modified to produce A method for producing a polypeptide of interest comprising:
(A) culturing a mutant of the parent Aspergillus cell according to any one of claims 1 to 10 , wherein (i) a first nucleic acid sequence encoding the polypeptide by the mutant And (ii) the mutant produces a reduced or undetectable level of cyclopiazonic acid compared to the parental Aspergillus cells cultured under the same conditions, and (b) the above Isolating the polypeptide from the culture medium;
Comprising a method. 11. Producing Aspergillus mutant host cells according to any one of claims 1 to 10 which produce reduced or undetectable levels of cyclopiazonic acid compared to parental Aspergillus cells cultured under the same conditions. A method comprising: genomic inactivation of a gene encoding dimethylallyl-cycloacetoacetyl-L-tryptophan synthase. The genome inactivation may include dimethylallyl by specific or random mutagenesis, PCR-generated mutagenesis, site-specific DNA deletion, insertion and / or substitution, gene disruption or gene replacement, antisense technology, or a combination thereof. The method according to claim 12, which is carried out by modifying a gene encoding cycloacetoacetyl-L-tryptophan synthase. The method according to claim 12 or 13 , wherein the genome inactivation is performed by partially or completely removing a gene encoding dimethylallyl-cycloacetoacetyl-L-tryptophan synthase from a cell.

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